The folic acid analogue amethopterin, N-[p-(2,4-diamino-6-pteridylmethyl)methylaminobenzoyl]-glutamic acid, has been used for about 25 years as an anti-neoplastic agent. The related compounds aminopterin, N-{p-[(2,4-diamino-6-pteridylmethyl)amino] benzoyl}-glutamic acid, and dichloro-methotrexate, N-[p-(2,4-diamino-3,5-dichloro-6-pteridylmethyl)methylaminobenzoyl]-glutam ic acid, are similarly employed. These compounds are highly toxic, and the concentration of the same in a particular patient's system can vary despite controlled dosages. Accordingly, it is essential that the concentration in the recipient's system of the particular compound being administered be known and readily determinable at all times during treatment. The present invention provides a simple, economical and rapid method for determining the concentration of methotrexate or its assay equivalents in biological material, especially serum and cerebrospinal fluid.
As indicated above, amethopterin (hereinafter referred to as methotrexate or MTX) is widely used, and the mechanism by which it acts in biological systems has been studied rather extensively as noted in Bertino, J. R., "The Mechanism Of Action Of The Folate Antagonists In Man," Cancer Research 23, pages 1286 - 1306, 1963. These prior art studies and related efforts have disclosed the binding of methotrexate by dihydrofolic acid reductase (hereafter referred to as folic acid reductase), and assay techniques wherein determinations of enzymatically reactive substances are made by monitoring changes in the reaction solution which are proportional to the reaction or amount of the enzyme to be determined. However, it is believed that no direct binding studies using radiolabeled methotrexate or the further compounds noted above have been done with folic acid reductase.
The binding reaction techniques of the present invention are to be distinguished from prior art enzyme determinations wherein the enzyme is not bound per se, but rather ascertained by indirect means such as competitive oxidation of a coreactant. The subject binding techniques are also distinguished from proteinantibody radioimmunoassay techniques which are directed to determinations of complex, high molecular weight protein molecules for which specific antibodies are typically obtained by animal immunizing procedures. In contrast, the subject techniques employ an enzyme binder preparation which forms a stable complex with the ligand to be determined in the presence of a cofactor. The use of an enzyme binder preparation is advantageous since it generally has a higher specificity, a higher affinity and it is more readily available as compared with antibody materials. Thus, the procedures of the present invention employ radiochemical ligand binding techniques to provide a rapid and sensitive assay.